IDENTIFICATION AND ANALYSIS OF A MATRIX-ATTACHMENT REGION-5' OF THE RAT GLUTAMATE-DEHYDROGENASE-ENCODING GENE

Eukaryotic chromatin is thought to be organized into independently reg ulated loop domains by interaction of matrix-attachment regions (MAR) of the DNA to the nuclear matrix. To define the borders of the chromat in loop containing the glutamate dehydrogenase (GDH) gene, we screened the GDH gene and flanking regions for the presence of MAR sequences. We here report identification, mapping and sequencing of an (A+T)-rich MAR located 2010-1397 bp upstream of the transcription initiation sit e of GDH, that mediates strong binding to the nuclear matrix. Smaller regions can also confer binding capacity, although at a lower affinity . This (A+T)-rich MAR contained 11 bp and 12 bp (A+T)-rich direct repe ats, but not any of the sequences previously described to be associate d with MAR activity. We here show that the presence of (A+T)-rich doma ins of DNA is not sufficient to confer binding capacity, since (A+T)-r ich sequences located downstream of the identified MAR did not bind to the nuclear matrix. Moreover, a consensus topoisomerase-II-binding si te located downstream of the MAR was found to be insufficient to media te substantial binding. The number of binding sites in the nuclear mat rix for MAR-containing fragments was shown to be approximately 15 000/ nucleus. Since organization of the entire rat genome in loops with an average loop size of 100 kbp would require 60 000 binding sites, this suggests that only part of the genome is organized in loops. Alternati vely, we might have underestimated the number of binding sites. The GD H MAR, and MAR-containing fragments derived from other species, were f ound to bind to the same binding sites in the nuclear matrix, although the affinity varied.